Liquid Formulations of Bendamustine

ABSTRACT

Stable liquid formulations of bendamustine, and pharmaceutically acceptable salts thereof, and polar aprotic solvents, are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/221,422, filed Mar. 21, 2014, which is a continuation of U.S.application Ser. No. 14/151,242, filed Jan. 9, 2014, which is acontinuation of U.S. application Ser. No. 13/655,498, filed Oct. 19,2012, which is a continuation of U.S. application Ser. No. 13/362,430,filed Jan. 31, 2012, now U.S. Pat. No. 8,344,006, which is acontinuation of U.S. application Ser. No. 13/048,325, filed Mar. 15,2011, now abandoned, which is a continuation of InternationalApplication No. PCT/US2009/58023, filed Sep. 23, 2009, which claims thebenefit of U.S. Provisional Application No. 61/100,074, filed Sep. 25,2008, the entireties of which are incorporated by reference herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to liquid formulations of bendamustine,and the pharmaceutical salts thereof.

BACKGROUND OF THE INVENTION

Bendamustine,(4-{5-[bis(2-chloroethyl)amino]-1-methyl-2-benzimidazolyl}butyric acid

is an atypical structure with a benzimidazole ring, which structureincludes an active nitrogen mustard. Bendamustine was initiallysynthesized in 1963 in the German Democratic Republic and was availablefrom 1971 to 1992 in that location under the name Cytostasan®. Sincethat time, it has been marketed in Germany under the tradenameRibomustin®. It is currently available for use in the United Statesunder the tradename Treanda® (Cephalon, Inc., Frazer, Pa.). It has beenwidely used to treat chronic lymphocytic leukemia, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma, and breast cancer.

Like other nitrogen mustards, bendamustine hydrolyzes in aqueoussolution, with the major degradant being the primary alcohol HP1 (SeeU.S. application Ser. No. 11/330,868, the entirety of which isincorporated herein):

In light of its instability in aqueous solution, bendamustine iscurrently supplied as a lyophilized powder for injection. Just prior toits infusion, the medical practitioner reconstitutes the powder withSterile Water for Injection. Reconstitution should yield a clear,colorless to pale yellow solution and the powder should completelydissolve in about 5 minutes. If particulate matter is observed, thereconstituted product should not be used and should be discarded. Thereconstituted product is then transferred to a 0.9% Sodium ChlorideInjection infusion bag within 30 minutes of reconstitution. Thisadmixture should be a clear and colorless to slightly yellow solution.If the admixture comprises particulate matter or is discolored, itshould be discarded and a fresh sample prepared.

The reconstitution of the bendamustine lyophilized powder is timeconsuming and cumbersome. Moreover, lyophilization of solids on acommercial scale requires specialized equipment and incurs significantexpense. As such, formulations of bendamustine that do not requirelyophilization and/or reconstitution are needed.

Solutions of bendamustine hydrochloride in anhydrous propylene glycol,prepared under an inert gas atmosphere, have been reported (GDR Patent159289). It was reported that analysis of these solutions usingthin-layer chromatography, eluting with butanol/acetic acid/water(4:1:5) and detection with Dragendorff reagent and UV (360 nm) did notsuggest any decomposition. Curiously, however, commercial development ofpropylene glycol formulations have heretofore not been reported. Thus,improved liquid formulations of bendamustine are still needed.

SUMMARY OF THE INVENTION

The present invention is directed to liquid pharmaceutical formulationscomprising bendamustine, or a pharmaceutically acceptable salt orprodrug thereof, and a polar aprotic solvent. Certain preferredembodiments include liquid pharmaceutical formulations comprisingbendamustine, or a pharmaceutically acceptable salt or prodrug thereof,a polar aprotic solvent, and a non-aqueous polar protic solvent. Methodsof making and using the formulations of the present invention are alsodescribed, as are methods of treating cancer using the claimedformulations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of a stability analysis of bendamustine in varioussolvents at 25° C.

FIG. 2 is a graph of a stability analysis of bendamustine in varioussolvents at 5° C.

FIG. 3 is a graph of bendamustine purity, over time, in 99% propyleneglycol, at 5° C. and at 25° C.

FIG. 4 shows the mean+standard deviation concentration-versus-timeprofiles of bendamustine in male Cynomolgus monkeys (N=4) administeredsingle 3 mg/kg bolus intravenous doses of bendamustine hydrochloride in3 different formulations.

DETAILED DESCRIPTION OF THE INVENTION

Stable, liquid formulations of bendamustine have been discovered and arereported herein.

Experiments to produce commercially viable propylene glycol preparationshave been performed. Unfortunately, the results described in GDR Patent159289 were not reproducible. Solutions of bendamustine in 99% propyleneglycol degraded to non-bendamustine products over a time equivalent tocommercial storage. Two of the impurities were identified as propyleneglycol esters of bendamustine. As such, a 100% propylene glycolcommercial formulation of bendamustine is not feasible forpharmaceutical purposes.

It has been determined that pharmaceutically acceptable liquidformulations of bendamustine, and the pharmaceutically acceptable saltsthereof, in particular the hydrochloride salt, can be prepared bycombining bendamustine, or the pharmaceutically acceptable salt thereof,with a polar aprotic solvent or mixture of polar aprotic solvents.Polar, aprotic solvents are known in the art and include, for example,1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone,dimethylacetamide, dimethyl sulfoxide, acetone, tetrahydrofuran,1,4-dioxane, acetonitrile, dimethyl formamide, propylene carbonate. Seealso, e.g., Florence Mottu, et al. Organic solvents for pharmaceuticalparenterals and embolic liquids: A review of toxicity data, PDA J.Pharma. Sci. & Tech. vol 54, no. 6, 456-469 (November-December 2000).Particularly preferred polar aprotic solvents include dimethylacetamide,dimethyl sulfoxide, and mixtures thereof.

Without wishing to be held to any particular theory, it is believed thatpolar, aprotic solvents are sufficiently non-nucleophilic towardsbendamustine such that polar aprotic solvent-bendamustine adducts do notform over the course of typical commercial storage conditions. Typicalcommercial storage conditions include time periods of, for example,about 30 days, about 90 days, about 180 days, and about 365 days (about1 month, about 3 months, about 6 months, and about 1 year). Typicalcommercial storage conditions also include temperatures of about 23° C.(ambient room temperature) and refrigerated temperatures below ambientroom temperature, for example, about 5° C. Preferably, the liquidformulations of the present invention are stored at refrigeratedtemperatures.

It has also been discovered that stable formulations of bendamustine canbe obtained by mixing a polar aprotic solvent, or a mixture of polaraprotic solvents, with a non-aqueous polar protic solvent or mixture ofnonaqueous polar protic solvents. Pharmaceutically acceptable nonaqueouspolar protic solvents are known in the art and include alkyl alcohols,for example, ethanol, ethylene glycol, propylene glycol, butyleneglycol, glycerin, polysorbates, for example TWEEN 20, TWEEN 40, andTWEEN 80, and cyclodextrins (such as hydroxypropyl-β-cyclodextrin),polyalkylene glycols, such as polyethylene glycol, polypropylene glycol,and polybutylene glycol, and primary amides such as niacinamide.

Such formulations will typically comprise 90% or less, by volume of theformulation, of the nonaqueous polar protic solvent. In other preferredembodiments, formulations will comprise between about 20% and about 85%,by volume of the formulation, of the nonaqueous polar protic solvent. Instill other embodiments, formulations will comprise between about 30%and about 70%, by volume of the formulation, of the nonaqueous polarprotic solvent. In most preferred embodiments, formulations willcomprise about 80%, about 67% or about 34%, by volume of theformulation, of the nonaqueous polar protic solvent.

Alternatively, formulations of the present invention will comprise 10moles per liter, or less, of the nonaqueous polar protic solvent.Preferably, formulations of the present invention will comprise betweenabout 4 moles per liter to about 9.5 moles per liter, of the nonaqueouspolar protic solvent. In certain embodiments, formulations will compriseabout 9.1 moles per liter of the nonaqueous polar protic solvent. Inother embodiments, formulations will comprise about 4.6 moles per liter,of the nonaqueous polar protic solvent.

While not wishing to be held to any particular theory, it is believedthat while nonaqueous polar protic solvents are of sufficientnucleophilicity to form potentially undesirable polar proticsolvent-bendamustine adducts, such adducts will not form during typicalcommercial storage if the concentration of the polar protic solvent iskept within the scope of the present invention.

Liquid formulations of the present invention are stable over the courseof a typical commercial storage period. As used herein, “stable” isdefined as no more than about a 10% loss of bendamustine under typicalcommercial storage conditions. Preferably, formulations of the presentinventions will have no more than about a 10% loss of bendamustine, morepreferably, no more than about a 5% loss of bendamustine, under typicalcommercial storage conditions.

Bendamustine converts to non-bendamustine products (i.e., “degrades”)upon exposure to certain nucleophiles, for example, water and alkyeneglycols such as propylene glycol. Exposure of bendamustine to water canproduce “HP1,” which is undesirable.

Another undesirable compound that bendamustine can convert to over timeis “BM1 dimer”

Still another undesirable compound that bendamustine can convert to overtime is “DCE.”

Upon exposure to an alkylene glycol, for example, propylene glycol,esters of bendamustine can form. e.g., PG-1 and PG-2.

In preferred embodiments of the present invention, analysis offormulations of the present invention will exhibit 1.50% or less of DCE,as determined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C. More preferably, the formulations will exhibit 1.0% or lessof DCE, as determined by HPLC analysis, after about 1 year (about 365days) at about 5° C. Even more preferably, the formulations will exhibit0.5% or less of DCE, as determined by HPLC analysis, after about 1 year(about 365 days) at about 5° C. Most preferably, the formulations willexhibit about 0.1% or less of DCE, as determined by HPLC analysis, afterabout 1 year (about 365 days) at about 5° C.

In other embodiments of the present invention, analysis of theformulations will exhibit about 0.4% or less of HP1, as determined byHPLC analysis, after about 1 year (about 365 days) at about 5° C.Preferably, the formulations will exhibit about 0.10% or less of HP1, asdetermined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C.

In certain other embodiments of the present invention, analysis of theformulations will exhibit about 0.70% or less of BM1 dimer, asdetermined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C. Preferably, the formulations will exhibit about 0.30% orless of dimer, as determined by HPLC analysis, after about 1 year (about365 days) at about 5° C. In most preferred embodiments, the formulationswill exhibit about 0.10% or less of BM1 dimer, as determined by HPLCanalysis, after about 1 year (about 365 days) at about 5° C.

In those embodiments of the present invention comprising alkylene glycolas the nonaqueous polar protic solvent, analysis of those formulationswill exhibit 1.5% or less of alkylene glycol esters of bendamustine, asdetermined by HPLC analysis, after about 1 year (about 365 days) atabout 5° C. For example, in those embodiments comprising propyleneglycol, analysis of those formulations will exhibit 1.5% or less ofpropylene glycol esters PG-1 and PG-2, as determined by HPLC analysis,after about 1 year (about 365 days) at about 5° C.

Analysis of the liquid formulations of the present invention can beperformed using techniques known in the art, including, for example,HPLC, gas chromatography, and NMR. After exposure to typical commercialstorage conditions, analysis of the formulations of the presentinvention will indicate that the formulation contains no less than about90% of the amount of bendamustine present prior to exposure to thestorage conditions. Preferably, analysis will indicate that theformulation contains no less than about 95% of the amount ofbendamustine present prior to exposure to the storage conditions.

In preferred embodiments of the present invention, analysis of theformulations of the present invention will indicate that the formulationcontains no less than about 90% of the amount of bendamustine presentprior to exposure to storage conditions that include temperatures ofabout 5° C. and time periods of about 30 days (about 1 month) to about365 days (about 1 year). Preferably, analysis of the formulations of thepresent invention will indicate that the formulation contains no lessthan about 90% of the amount of bendamustine present prior to exposureto storage conditions that include temperatures of about 5° C. and timeperiods of about 30 days (about 1 month), about 90 days (about 3months), and about 180 days (about 6 months). Preferably, analysis willindicate that the formulation contains no less than about 95% of theamount of bendamustine present prior to exposure to storage conditionsthat include temperatures of about 5° C. and time periods of about 30days (about 1 month) to about 365 days (about 1 year). More preferably,analysis will indicate that the formulation contains no less than about95% of the amount of bendamustine present prior to exposure to storageconditions that include temperatures of about 5° C. and time periods ofabout 30 days (about 1 month), about 90 days (about 3 months), and about180 days (about 6 months).

Formulations of the present invention can comprise pharmaceuticallyuseful concentrations of bendamustine, or a pharmaceutically acceptablesalt thereof. Useful concentrations include concentrations ranging fromabout 5 mg/mL to about 200 mg/mL. Preferably, the concentration ofbendamustine, or a pharmaceutically acceptable salt thereof, ranges fromabout 5 mg/mL to about 120 mg/mL. Preferred concentrations include about5 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL,about 50 mg/mL, about 60 mg/mL, about 100 mg/mL and about 200 mg/mL ofbendamustine, or a pharmaceutically acceptable salt thereof. Greaterthan 200 mg/ml of bendamustine, or a pharmaceutically acceptable saltthereof, for example, greater than about 300 mg/mL, are also within thescope of the present invention, as are saturated solutions ofbendamustine, or a pharmaceutically acceptable salt thereof.

As used herein, the term “about” is defined as ±10%, preferably ±5%,

In addition to comprising a polar aprotic solvent, or mixture of polaraprotic solvents, and optionally, a nonaqueous polar protic solvent, ormixture of solvents, formulations of the present invention may furthercomprise other pharmaceutically acceptable excipients. Pharmaceuticallyacceptable excipients are known in the art and include, for example,antioxidants (e.g., tocopherol (Vitamin E), ascorbic acid, methylparaben, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), andpropyl gallate), surfactants, (e.g., polysorbates (TWEEN 20, TWEEN 40,TWEEN 80)), lipids (e.g., dimyristoylphophatidylcholine (DMPC),Dimyristoylphosphatidylglycerol (DMPG), distearoylphophatidylglycerol(DSPG), fillers (e.g., mannitol), organic acids (e.g., citric acid,lactic acid, benzoic acid), hydrophilic polymers (e.g., polyethyleneglycols (PEG 300, PEG 400), complexing agents (e.g., niacinamide,nicotinic acid, creatine, cyclodextrins), and preservatives (e.g.,benzyl alcohol).

Also within the scope of the invention are methods of treating diseases,such as, for example, chronic lymphocytic leukemia, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma, or breast cancer, with apharmaceutical formulation of the present invention. These methodscomprise administering to the patient a therapeutically effective amountof a preparation prepared from a pharmaceutical formulation of thepresent invention. The term “therapeutically effective amount,” as usedherein, refers to the amount determined to be required to produce thephysiological effect intended and associated with a given drug, asmeasured according to established pharmacokinetic methods andtechniques, for the given administration route. Appropriate and specifictherapeutically effective amounts can be readily determined by theattending diagnostician, as one skilled in the art, by the use ofconventional techniques. The effective dose will vary depending upon anumber of factors, including the type and extent of progression of thedisease or disorder, the overall health status of the particularpatient, the relative biological efficacy of the compound selected, theformulation of the active agent with appropriate excipients, and theroute of administration.

The liquid formulations of bendamustine described herein are intended tobe administered via injection, for example, they may be administeredsubcutaneously, intracutaneously, intravenously, intramuscularly,intra-articularly, intrasynovially, intrasternally, intrathecally,intralesionally, intracranially or via infusion. In a typicalpreparation, the volume of the liquid formulation of the presentinvention needed for the required dose can be aseptically withdrawn andtransferred to an infusion bag of 0.9% Sodium Chloride (or otherpharmaceutically acceptable intravenous solution) for injection. Aftertransfer, the contents of the infusion bag are thoroughly mixed.Administration by intravenous infusion is typically provided over a timeperiod of from about 30 to about 60 minutes. Previously describedlyophilized formulations of bendamustine required reconstitution of thelyophilized bendamustine prior to mixture with the acceptableintravenous solution before infusion.

It is envisioned that the pharmaceutical formulations and preparationsof the present invention can be administered in combination with one ormore anti-neoplastic agents where the anti-neoplastic agent is givenprior to, concurrently with, or subsequent to the administration of theformulation or preparation of the present invention. Pharmaceuticallyacceptable anti-neoplastic agents are known in the art. Preferredanti-neoplastic agents are those disclosed in co-pending U.S.application Ser. No. 11/330,868, filed Jan. 12, 2006, the entirety ofwhich is incorporated herein by reference.

Examples Solubility and Stability of Bendamustine Hydrochloride in PolarAprotic Solvents

Equilibrium solubility was determined for solvents including1-methyl-2-pyrrolidone (NMP), 1,3-dimethyl-2-imidazolidinone (DMI),dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetone,tetrahydrofuran (THF), dimethylformamide (DMF), and propylene carbonate(PC). The solubility of bendamustine hydrochloride was also determinedfor two solutions, 25 mg/mL niacinamide in DMA and 66% DMA/34% propyleneglycol (PG). A saturated solution of bendamustine hydrochloride was madein triplicate for each solvent or solution and mixed on a Lab-Quake withgentle mixing and low shear for 3 days at room temperature. A sample ofeach suspension was put into a microcentrifuge tube and spun at 10,000rpm for 5 min on an Eppendorf microcentrifuge. The supernatant wasremoved and put into a clean vial. Each solution was diluted with samplesolvent: 50% NMP/50% 0.1% trifluoroacetic acid in water. A reverse phasemethod for bendamustine hydrochloride was used to determine theconcentration of each sample calculated from a standard. Analysis wasperformed within 18 hours of preparation of the diluted sample. Thesolubilities are listed in Table I below. Each value is an average ofthree samples.

TABLE I Sample* % Purity Assay (mg/mL) NMP 99.1 104.0 DMI 98.5 75.8 DMSO99.5 311.7 DMF 99.6 71.8 66% DMA/34% PG 99.5 110.1 DMA 99.4 56.2 PC 98.77.7 Niacinamide/DMA 99.2 61.3 *acetone and THF have no measurablesolubility of bendamustine.

The three replicates were combined and mixed well and then pipetted intoamber HPLC vials and placed in stability chambers at 25° C. and 5° C.All the samples were clear and colorless except for the DMI sample whichwas clear and yellow. The 25° C. stability leveled out from about 180days (about 6 months) to about 365 days (about 12 months, about 1 year).At 5° C., all solutions had a purity greater than 90%. The analysis ofstability samples can be seen in the graphs of FIGS. 1 and 2.

TABLE II Impurity profile of certain liquid formulations of BendamustineHCl after storage at 5° C. for about 12 months BM1 DCE HP1 dimer PG-1PG-2 Formulation (Area %) (Area %) (Area %) (Area %) (Area %)Niacinamide/ 1.40 0.08 0.06 ND ND DMA DMA 1.10 0.08 0.05 ND ND 66% DMA/0.12 0.08 0.06 1.09 0.27 34% PG DMF 0.07 0.11 0.07 ND ND NMP 0.90 0.10ND ND ND DMSO 0.04 0.38 0.70 ND ND ND = not detectedAnalysis conducted using reverse phase HPLC with 50% NMP/50% 0.1%trifluoroacetic acid in water as the running solvent.

As can be seen in FIG. 3, bendamustine (BM1) in 99% propylene glycoldegrades significantly when stored at 25° C. for less than 100 days.After storage at 5° C. for about 365 days, the purity of thebendamustine is about 80% or less.

Pharmacokinetic Study of Formulations in Monkey

4 fasted (18 to 23 hr), drug-naive male cynomolgus monkeys consecutivelyreceived single 3-mg/kg bolus intravenous doses of bendamustinehydrochloride prepared from 3 different formulations. The formulationsevaluated in the study included: 1) TREANDA (lyophilized mixture ofbendamustine hydrochloride and mannitol; 25 mg (bendamustinehydrochloride) vials; 2) a 66% dimethylacetamide (DMA)/34% propyleneglycol (PG) (w/w) solution (90 mg (bendamustine hydrochloride)/mLstock); and 3) a 100% DMA solution (45 mg (bendamustinehydrochloride)/mL stock). The lyophilized powder and stock solutions ofbendamustine hydrochloride were constituted or diluted with 0.9% saline,as appropriate, to give solutions of 3 mg bendamustine hydrochloride/ml,just prior to dose administration. The resulting solutions wereadministered as a bolus via a saphenous vein at a fixed volume of 1.0mL/kg. There was at least a 7-day washout period separating successivedoses. During all 3 phases of dosing, blood samples for pharmacokineticprofiling of bendamustine and its 2 active circulating metabolites,γ-hydroxybendamustine (M3) and N-des-methylbendamustine (M4), werecollected via a femoral vein immediately prior to dosing and atpreselected timepoints through 12 hr postdose. Concentrations ofbendamustine, M3 and M4 in plasma samples were determined using avalidated high-performance liquid chromatography method with tandem massspectrometric detection (LC-MS/MS) as follows. Bendamustine and the M3and M4 metabolites are extracted from plasma by protein precipitationusing acetonitrile. After the extraction, the aliquoted sample isacidified with 1% formic acid and bendamustine with an added carbon inthe carboxylic acid chain is added as an internal standard. The samplesare evaporated to dryness and the residue is reconstituted with anacetonitrile/water/formic acid/ammonium formate mixture. The sample isinjected into an HPLC system with LC/MS/MS detection using a PhenomenexSynergi Max-RP column with an acetonitrile/water/formic acid/ammoniumformate mobile phase. Pharmacokinetic analyses were performed usingnoncompartmental methods.

After single bolus intravenous doses of bendamustine hydrochloride tomale cynomolgus monkeys, the shapes of the mean plasmaconcentration-versus-time profiles of bendamustine were similar in eachof the 3 formulations (See FIG. 4). In all cases, the highest observedplasma levels of bendamustine were achieved at 0.083 hr postdose (ie,the first sampling time after dose administration) and subsequentremoval of the compound from plasma occurred in a bi-phasic manner thatwas characterized by an initial rapid distribution phase and a somewhatslower terminal phase of drug elimination. The harmonic mean t_(1/2) ofthe terminal phase was approximately 0.6 hr for each formulation (SeeTable III).

In addition to the similarities in the shapes of the mean plasmaconcentration-versus-time profiles, the 3 formulations were also similarwith respect to bendamustine systemic exposure (i.e., C_(max) and AUC).Specifically, the respective mean values of C_(max) and AUC_(0-∞) forbendamustine were 6037 ng/mL and 2314 ng·hr/mL for the TREANDAformulation, 7380 ng/mL and 2854 ng·hr/mL for the 66% DMA/34% PGformulation and 6209 ng/mL and 2372 ng·hr/mL for the 100% DMAformulation. Plasma clearance (CL) and volume of distribution (V_(z) andV_(ss)) for bendamustine were also comparable between each of the 3formulations (See Table III). In Table III, t_(max), hr is given asMedian [range], t_(1/2), hr is given as the Harmonic Mean, λ_(z), hr⁻¹is the slope of line in elimination phase used to calculate half-life,and MRT_(0-∞) is the mean residence time.

In summary, the pharmacokinetic profiles of bendamustine, M3 and M4 forthe 2 liquid formulations of bendamustine hydrochloride werequalitatively and quantitatively similar to those obtained for theTREANDA formulation after single bolus intravenous doses to monkeys.

Table III shows the mean+/−Standard Deviation pharmacokinetic parametersof bendamustine in male Cynomolgus monkeys (N=4) administered single 3mg/kg bolus intravenous doses of bendamustine hydrochloride in the threedifferent formulations.

TABLE III Formulation 66% DMA/ Parameter TREANDA 34% PG 100% DMAC_(0,)ng/mL 8664 ± 3841 10716 ± 2033  8956 ± 1965 C_(max), ng/mL 6037 ±2456 7380 ± 1170 6209 ± 1300 t_(max), hr 0.083 0.083 0.083 [0.083 forall] [0.083 for all] [0.083 for all] AUC_(0-t), ng · hr/mL 2313 ± 800 2853 ± 398  2371 ± 535  AUC_(0-∞), ng · hr/mL 2314 ± 800  2854 ± 398 2372 ± 535  λ_(z), hr⁻¹ 1.220 ± 0.111 1.295 ± 0.108 1.092 ± 0.219t_(1/2), hr 0.57  0.54  0.63  CL, L/hr/kg 1.27 ± 0.40 0.96 ± 0.14 1.18 ±0.27 V_(z), L/kg 1.04 ± 0.36 0.74 ± 0.05 1.17 ± 0.44 V_(ss), L/kg 0.34 ±0.11 0.26 ± 0.05 0.30 ± 0.04 MRT_(0-∞), hr 0.26 ± 0.02 0.27 ± 0.02 0.26± 0.03

In-Use Studies of Formulations

Admixtures in 0.9% sodium chloride (500 mL bag) were prepared at a highdose (360 mg bendamustine hydrochloride) and purity was determined overtime at room temperature for up to 8 hours using HPLC, using a ZorbaxBonus-RP column with a gradient from 93% 0.1% trifluoroacetic acid inwater (Mobile Phase A)/7% 0.1% trifluoroacetic acid in acetonitrile(Mobile Phase B) to 10% Mobile Phase A/90% Mobile Phase B.

The 66% DMA/34% PG formulation had a concentration of bendamustinehydrochloride of 90 mg/g, so 4 mL was injected into a 500 mL bag ofsaline, inverted 10 times and sampled at room temperature for 8 hours.After 8 hours the purity was 95.4%. This is within the labelrequirements for dosing Treanda. This formulation of the presentinvention could be used for up to 8 hours at room temperature. By way ofcontrast, reconstituted Treanda can only be stored at room temperaturefor up to 3 hours.

The 100% DMA formulation had a concentration of 45 mg/g, so 8 mL wasinjected into a 500 mL bag of saline, inverted 10 times, and sampled atroom temperature for 4 hours. After 4 hours the purity was 97.9%. Thisformulation of the present invention could be used for more than 4 hoursat room temperature.

The comparative Treanda admixture purity was 95.0% after 4 hours at 25°C.

As those skilled in the art will appreciate, numerous modifications andvariations of the present invention are possible in view of the aboveteachings. It is therefore understood that within the scope of theappended claims, the invention can be practiced otherwise than asspecifically described herein, and the scope of the invention isintended to encompass all such variations.

1. A stable, non-aqueous, liquid formulation comprising from about 5mg/mL to about 200 mg/mL of bendamustine or a pharmaceuticallyacceptable salt thereof, and up to about 90% of a mixture ofpolyethylene glycol and propylene glycol wherein said formulationcontains not less than 95% of the amount of bendamustine present priorto exposure to storage conditions that include a temperature of about 5°C. and a time period of about 180 days.
 2. The liquid formulation ofclaim 1, further comprising an antioxidant.
 3. The liquid formulation ofclaim 1, wherein said formulation comprises 1.5% or less of PG-1, PG-2,or a combination thereof

as determined by HPLC analysis, after about 1 year of storage at about5° C.
 4. A method of treating cancer comprising: providing the stable,non-aqueous, liquid formulation of claim 1; diluting the stable,non-aqueous, liquid formulation with a pharmaceutically acceptableinjectable diluent to form an injectable pharmaceutical preparation;administering the injectable pharmaceutical preparation to a patient inneed of treatment for cancer.
 5. The method of claim 4, wherein thecancer is chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin'slymphoma, multiple myeloma, or breast cancer.
 6. The method of claim 4,wherein the cancer is chronic lymphocytic leukemia or non-Hodgkin'slymphoma.
 7. An anhydrous, sterile, liquid, pharmaceutical formulationof bendamustine, or a pharmaceutically acceptable salt thereof,comprising propylene glycol and 1.5% or less of PG-1, PG-2, or acombination thereof

as determined by HPLC analysis, wherein the analysis is performed afterabout 1 year of storage.
 8. The anhydrous, liquid, pharmaceuticalformulation of claim 7, wherein the analysis is performed after about 1year of storage at about 5° C.
 9. The anhydrous, liquid, pharmaceuticalformulation of claim 7, comprising at least about 90% of the amount ofbendamustine present prior to storage of about 1 year, as determined byHPLC analysis.
 10. The anhydrous, liquid, pharmaceutical formulation ofclaim 9, wherein the storage is at about 5° C.
 11. The anhydrous,liquid, pharmaceutical formulation of claim 7, comprising at least about90% of the amount of bendamustine present prior to storage of about 180days, as determined by HPLC analysis.
 12. The anhydrous, liquid,pharmaceutical formulation of claim 11, wherein the storage is at about5° C.
 13. The anhydrous, liquid, pharmaceutical formulation of claim 7,comprising at least about 90% of the amount of bendamustine presentprior to storage of about 90 days, as determined by HPLC analysis. 14.The anhydrous, liquid, pharmaceutical formulation of claim 13, whereinthe storage is at about 5° C.
 15. The anhydrous, liquid, pharmaceuticalformulation of claim 7, comprising at least about 90% of the amount ofbendamustine present prior to storage of about 30 days, as determined byHPLC analysis.
 16. The anhydrous, liquid, pharmaceutical formulation ofclaim 15, wherein the storage is at about 5° C.
 17. The anhydrous,liquid, pharmaceutical formulation of claim 7, comprising bendamustineat a concentration of about 5 mg/mL to about 200 mg/mL.
 18. A method oftreating cancer comprising providing the anhydrous, liquid,pharmaceutical formulation of claim 7; diluting the anhydrous, liquid,pharmaceutical formulation with a pharmaceutically acceptable injectablediluent to form an injectable pharmaceutical preparation; administeringthe injectable pharmaceutical preparation to a patient in need oftreatment for cancer.
 19. The method of claim 18, wherein the cancer ischronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma,multiple myeloma, or breast cancer.
 20. The method of claim 18, whereinthe cancer is chronic lymphocytic leukemia or non-Hodgkin's lymphoma.